Nanoparticles having a size of the order of 2-100 nm in particular are the focus of increasing intention for applications in imaging and diagnosis since they offer a very large specific surface area, the increase in the surface area of a sphere being proportional to the square of its radius. In addition, for in vivo applications the mechanisms of diffusion in tissues and the cell entry of nanoparticles differ from those of objects of larger size which are rapidly eliminated by the body. For such applications it is often necessary for these nanoparticles to be carriers of oligonucleotides or peptides. Within this context, the inventors of the present patent application have turned to the issue of preparing nanoparticles as support for conducting solid-phase synthesis requiring chain reactions, as is the case in particular for the synthesis of oligonucleotides or peptides.
It is difficult to couple peptides or oligonucleotides onto nanoparticles since the latter are difficult to sediment at the bottom of a tube (by centrifugation). Their replacing in solution is also very difficult and may give rise to problems of irreversible aggregation. In addition, the conformations (or three-dimensional structures) taken on by biomolecules in solution frequently cause strong steric hindrance around the reactive functions required for grafting onto nanoparticles. On this account, the coupling yields of biomolecules on objects of nanometric size are low. In the literature the highest grafting densities of biomolecules obtained using coupling techniques are of the order of 0.01-0.05 oligonucleotides/nm2 (Y. Cheng, T. Stakenborg, P. V. Dorpe, L. Lagae, M. Wang, H. Chen, G. Borghs, Anal Chem, 83 (2011) 1307-1314).
Additionally, automated solid-phase synthesis cannot be envisaged at the current time on nanoparticles since this strategy requires confinement of the nanoparticles in synthesis cells closed by porous filters of sintered type or porous diaphragms which raises two major problems. First, the minimum size of the pores of porous filters required so that the synthesis solvents and reagents are able to enter the synthesis cell without modifying the pressure of the circuit in which the solvents and reagents circulate (in general no more than 4 to 6 bars) is 2 to 4 μm. A smaller pore size for the porous filters would generate an increase in pressure leading to slower flow rates, to fouling of the surface of the support materials and hence to synthesis of poor quality. Also, there does not exist a porous filter which is both resistant to the organic solvents used for such synthesis (which are generally acetonitrile, dichloromethane, pyridine, tetrahydrofuran, . . . ) and which has a pore size of less than one micron.
In an endeavour to solve this issue, some of the inventors of the present patent application have proposed confining the nanoparticles by immobilising them temporarily on silica microparticles. Therefore, the inventors have already proposed assemblies called NOMs (Nano-on-Micro) formed of silica particles of micrometric size (2 to 4 μm) and of spherical shape coated with a monolayer of nanoparticles which offer an overall size of more than 4 μm, a size that is theoretically compatible with the strategy of automated solid-phase synthesis. Syntheses of fragments of deoxyribonucleic acids (DNA) have been successfully conducted (Farre et al., Langmuir, 2010, 26(7), 4941-4950, C. Farre, M. Lansalot, R. Bazzi, S. Roux, C. A. Marquette, G. Catanante, L. J. Blum, N. Charvet, C. Louis, C. Chaix, Langmuir, (2010) 26(7) 4941-4950).
Nevertheless, the proposed NOMs do not allow some difficulties to be overcome. First, the NOMs still of size that is too small, have a tendency to clog the filters and cannot properly be replaced in suspension between the synthesis steps. In addition, the mechanical friction applied to the NOMs by the flows of argon and the passing of the different reagents and solvents used for synthesis (which strongly agitate the particles) leads to the detachment of a large number of nanoparticles during synthesis. For example, it has been observed by some of the inventors that there is loss of nanoparticles during synthesis as well as poor quality of oligonucleotide syntheses on the nanoparticles.